Use of cationic microbubbles targeted to P-selectin to improve ultrasound-mediated gene transfection of hVEGF_(165) to the ischemic myocardium

超声靶向击碎微泡技术介导P-选择素靶向阳离子超声微泡改善人血管内皮生长因子165基因转染缺血心肌的实验性研究（英文）

Gene therapies have been applied to the treatment of cardiovascular disease, but their use is limited by the need to deliver them to the right target. We have employed targeted contrast ultrasound-mediated gene transfection （TCUMGT） via ultrasound-targeted microbubble destruction （UTMD） to transfer therapeutic genes to specific anatomic and pathological targets. Phospholipid microbubbles （MBs） with pcDNA3.l-human vascular endothelial growth factor 165 （pcDNA3.I-hVEGFls5） plasmids targeted to P-selectin （MB＋P＋VEGFp） were created by conjugating monoclonal antibodies against P-selectin to the lipid shell. These microbubbles were divided into four groups： microbubble only （MB）, microbubble＋P-selectin （MB＋P）, microbubble＋pcDNA3.l-hVEGF185 plasmid （MB＋VEGFp）, and microbubbie＋ P-selectin＋pcDNA3.1-hVEGF185 piasmid （MB＋P＋VEGFp）. The reverse transcription polymerase chain reaction （RT-PCR） and enzyme-linked immunosorbent assay （ELISA） results showed that the VEGF gene was successfully transfected by TCUMGT and the efficiency is increased with P-selectin targeting moiety. UTMD-mediated delivery of VEGF increased myocardial vascular density and improved cardiac function, and MB＋P＋VEGFp delivery showed greater improvement than MB＋VEGFp. This study drew support from TCUGMT technology and took advantage of targeted ultrasound contrast agent to identify ischemic myocardium, release pcDNA3.1-hVEGF165 recombinant plasmid, and improve the myocardial microenvironment, so promoting the restoration of myocardial function.

目的:构建一种靶向阳离子微泡,探讨其提高超声靶向击碎微泡技术(TCUMGT)介导的体内基因转染效率及治疗效果。创新点:提出运用TCUMGT介导的基因转染技术,利用微泡的携基因和靶向定位释放双项功能来上调缺血区的人血管内皮生长因子165(hVEGF_(165))的表达水平,发挥其成血管作用,从而改变缺血心肌的存活性。方法:通过聚乙二醇40硬脂酸酯、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、pcDNA_(3.1)-hV EGF165和抗P-选择素单克隆抗体等制备P-选择素靶向阳离子微泡。微泡分成四组:(1)仅微泡(MB);(2)微泡+P-选择素(MB+P);(3)微泡+pcDNA_(3.1)-hVEGF_(165)质粒(MB+VEGFp);(4)微泡+P-选择素+pcDNA_(3.1)-hVEGF_(165)质粒(MB+P+VEGFp)。逆转录聚合酶链反应(RT-PCR)和酶联免疫吸附试验(ELISA)结果显示:TCUMGT成功转染hVEGF_(165)基因,并且通过P-选择素为靶点可以提高转染效率。另外与其他组相比,MB+P+VEGFp组的心肌血管密度增加和心功改善最为明显。结论:本研究表明,通过TCUMGT技术,靶向超声微泡可以有效识别缺血心肌,释放pcDNA_(3.1)-hVEGF_(165)重组质粒,提高心肌微环境,促进心肌功能的恢复。